The goal of our current and proposed work is two fold. First,we want to understand how contractile protein muscle genes function in muscle and nonmuscle cells and how they are coordinately regulated during terminal differentiation of muscle cells. Our second goal is to identify and characterize the genes that initiate and regulate the myogenic program leading to terminal muscle cell differentiation. Towards these ends, we have focused on characterizing the structure and expression of the Drosophila tropomyosin genes, one of the more abundant proteins of the contractile apparatus. These genes exhibit both temporal and tissue-specific muscle expression and ubiquitous expression in nonmuscle cells. During the next granting period we will continue our current work to dissect the cis- and trans-regulatory apparatus regulating the temporal and muscle-specific expression of the tropomyosin I (TmI) gene. These experiments will consist of making deletion mutations of TmI-reporter chimeric fusion genes and studying their expression in P-element mediated germline transformed flies and in transiently expressing embryos and larvae. Using this same approach, we will continue our analysis of the muscle and cytoskeletal promoters of the tropomyosin II (TMII) gene. We will identify and characterize trans-acting protein factors that interact with the TmI cis-regulatory apparatus with specific emphasis on those factors that may coordinately regulate expression with the TmII and other contractile protein genes. Finally, we will use multiple approaches to identify cDNA clones expressed during early stages of muscle development that encode upstream factors involved in the regulation of the myogenic program. These experiments include cross-hybridization experiments with heterologous vertebrate gene probes, subtractive hybridization screening to identify myoblast-specific cDNA clones, and the analysis of a recently identified Drosophila muscle segment homeobox gene. By the end of this five-year granting period, we expect to have dissected the cis-acting control regions that regulate expression of the TmI, TmII and cytoskeletal Tm gene promoters. We plan to have identified and characterized trans-acting factors that are involved in regulating expression of these genes and to have begun cloning the corresponding cDNAs and genes. We expect to have cloned cDNAs encoding factors that regulate the myogenic program and to have begun an analysis of their function.